cell-preparation-for-flow-cytometry.pdfVIP免费

FlowCytometry–BestProtocols®Page1of3CellPreparationforFlowCytometryResearchUseOnlyRevised07-30-2013ProvidedasacourtesybyeBioscience,AnAffymetrixCompany•Copyright©2000-2013eBioscience,Inc.Tel:888.999.1371or858.642.2058•Fax:858.642.2046•www.ebioscience.com•info@ebioscience.comProtocolA:CellPreparationofTissueCultureCellsProtocolB:CellPreparationfromLymphoidTissueProtocolC:CellPreparationfromNon-LymphoidTissueProtocolD:IsolationofPBMCfromwholebloodIntroductionThesearethestandardprotocolsusedateBioscienceforthepreparationofvariouscelltypestobeusedforflowcytometricanalysis.Thisinformationistoserveasaguideasindividualinvestigatorsmayneedtooptimizeprotocolsfortheirparticularcelltype.GeneralNotes1.Singlecellsuspensionsarerequiredforoptimalstainingofsamplesforflowcytometry.2.Thenarrowboresofthesampleinjectionneedleandtubingonaflowcytometerwillbeeasilycloggedbyaggregatedcellsanddebris.3.Preparationofsinglecellsuspensionsfromsolidtissuerequiresmechanicaldissociationand/orenzymaticdigestionforoptimalrecoveryofcellsfromthetissue.Conditionsandenzymerequirementsfordigestionofthetissueofinterestwillneedtobedeterminedempirically.UsefulwebsitesWorthingtonTissueDissociationGuide(http://www.tissuedissociation.com)TheWorthingtonTissueDissociationGuideprovidesausefulsummaryandguideofthevariousmethodsthatcanbeusedfortissuedissociation.ProtocolA:CellPreparationofTissueCultureCellsMaterialsAccutase™EnzymeCellDetachmentMedium(Cat.No.00-4555)ortrypsinorEDTAeBioscience®FlowCytometryStainingBuffer(Cat.No.00-4222)15or50mLconicalcentrifugetubesExperimentalProcedure1.Forcellsthatgrowinsuspension,decantthecellsintoaconicalcentrifugetubeandperformacellcountandviabilityanalysis.ProceedtoStep4.2.Foradherentcellslines,detachyourcellsfromtheplate.Themostcommonmethodisscrapingbutcanresultincellclumpswhichcanclogthecytometer.Trypsinisanotheroption,butitcandestroytheepitopeoftheproteinyoumaybeinterestedinstaining.Youwillneedtotestthisempirically.EDTA(10mMinPBS)willdetachcellsandwi...